Identification of anti-hiv compounds inhibiting virus assembly and binding of nucleocapsid protein to nucleic acid

ABSTRACT

The invention provides methods and pharmaceutical compositions for inhibiting viral replication, particularly retroviral replication. The methods comprise administration of stibonic acid or diphenyl compounds that disrupt viral nucleocapsid binding to nucleic acids.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No.60/416,854, filed Oct. 8, 2002, which is herein incorporated byreference.

BACKGROUND OF THE INVENTION

It is well known that, under selection pressure, viruses often mutate todrug-resistant strains, thereby limiting the efficacy of most antiviralagents. Those viral structures that are required for viability andgrowth are often good drug targets because their inactivation cannot beeasily overcome by mutation. The utility of these targets can be furtherenhanced if the structures are mutationally intolerant. Furthermore,these structures may be conserved and/or maintained between virusfamilies, groups or genuses.

In particular retroviruses such as HIV, can become rapidly resistant todrugs used to treat the infection due to the high error rate of thereverse transcriptase enzyme responsible for transcribing its RNAgenome. HIV is an example of such a hyper-mutable virus. It has divergedinto two major species, HIV-1 and HIV-2, each of which has many clades,subtypes and drug resistant variations.

Strategies for coping with emergence of viral drug-resistant strainsinclude combination drug therapies (Lange (1996) AIDS 10 Suppl1:S27-S30). Drugs against different viral proteins and drugs againstmultiple sites on the same protein are commonly used as a strategy toovercome the adaptability of the virus. Combination therapies forretroviruses, using, e.g., protease inhibitors and nucleoside analogues,such as AZT, ddI, ddC and d4T, can become ineffectual; the virusdevelops complete resistance in a relatively short period of time (Birch(1998) AIDS 12:680-681; Roberts (1998) AIDS 12:453-460; Yang (1997)Leukemia 11 Suppl 3:89-92; Demeter (I 997) J. Acquir. Immune Defic.Syndr. Hum. Retrovirol. 14(2):136-144; Kuritzkes (1996) AIDS 10 Suppl5:S27-S31). Furthermore, no effective anti-retroviral vaccine iscurrently available (Bolognesi (1998) Nature 391:638-639; Bangham (1997)Lancet 350:1617-1621).

The HIV-1 caused AIDS epidemic began about 18 years ago. Since then thenumber of new cases have increased over time. By the end of 1994,1,025,073 AIDS cases had been reported to the WHO, with a 20% increasein the number of cases since December, 1993 (Galli (1995) Q. J. Nucl.Med. 39:147-155). By the year 2000, the WHO predicts that there will be30 to 40 million cumulative HIV-1 infections in the world (Stoneburner(1994) Acta Paediatr. Suppl. 400:1-4).

The Gag and Gag-Pol proteins in the Retroviridae, except forSpumaviruses, contain a highly conserved zinc finger motif (CCHC) withinthe nucleocapsid p7 (NCp7) protein portion of the polyprotein (seedefinitions, below). The absolute conservation of the metal chelatingcysteine and histidine residues along with other residues of the proteinand its in participation in essential functions during early and latevirus replication has identified this feature as an antiviral target.Mutations of the chelating residues in the zinc fingers yield anon-infectious virus. Because zinc fingers are identical in mostretroviruses, reagents able to inhibit its function have the potentialof being broad spectrum anti-viral therapeutic drugs. For example, ithas been shown that compounds that target the zinc finger byirreversible binding and cause ejection of the zinc molecule exhibitantiviral activity (see, e.g., U.S. Pat. No. 6,001,555; Rice et al.,Nature 361:473-475, 1993). Disulfide benzamidines were also shown to beactive in acutely and chronically infected cell lines (Rice et al.,Science 270:1194-1197, 1995), and a series of pyridinioalkanoylthioesters were developed that had superior anti-HIV-1 activity and lesstoxicity compared to the disulfide benzidines (see, e.g., Turpin et al.,J. Med. Chem. 42:67-86, 1999). A cyclic peptide that mimics severalbinding determinants in NC-p7 and inhibits NC-p7 annealing activitieshas also been designed (e.g., Druillennec et al., Proc Natl. Acad. SciUSA 96:4886-4891, 1999; Druillennec et al., Bioorg Med Chem Lett9:627-632, 1999). More recently, a series of tricyclic compounds havebeen identified that inhibit binding of NC-p7 to a shortoligonucleotide, d(TG)₄, and that have anti-HIV activity (see, e.g.,Stephen et al., Biochem. Biophys. Res. Comm. 296:1228-1237, 2002; and WO02/062333).

Antimony-containing oxo-metalate complexes have been disclosed that haveanti-viral activity (e.g., U.S. Pat. No. 5,041,576), however thesecompounds are limited to antimony oxo-metalate complexes that havemolybdate and tungstate moieties. The present invention now provides newmethods of inhibiting viral replication using antimony compounds ordiphenyl compounds that disrupt nucleocapsid/nucleic acid bindinginteractions.

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting viral replication byadministration of compounds that disrupt nucleocapsid binding to nucleicacids.

In one aspect, the invention provides a method of inhibiting replicationof a virus, said method comprising:

contacting a nucleocapsid protein of the virus with a compound havingthe formula:

wherein R¹⁴, R¹⁵ and R¹⁶ are members independently selected from H, NO₂,Sb(O)(OH)₂, OR⁷, SR¹⁷, CN, NR¹⁷R¹⁸, COR¹⁸, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl; wherein R¹⁷ and R¹⁸are members independently selected from H, OR¹⁹, C(O)R¹⁹, and NR¹⁹R²⁰;wherein R¹⁹ and R²⁰ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl, with the proviso that at least one of R¹⁴, R¹⁵ and R¹⁶ isother than H.

In one embodiment, at least one of R¹⁴, R¹⁵ and R¹⁶ comprises a memberselected from carboxylic acid, carboxylic acid ester, and carboxylicacid amide.

The invention also provides a method of inhibiting replication of avirus, the method comprising:

contacting a nucleocapsid protein of the virus with a compound havingthe formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, CN, OR¹¹, COR¹², NR¹¹R¹³, andCONR¹¹R¹³; wherein R¹¹ and R¹³ are members independently selected fromH, substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl; R¹² is a member selected from H, and OR¹³; and X is amember selected from O, S, and NR^(x); wherein R^(x) is a memberselected from H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl, with the proviso that at least three of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are COOR¹³.

In one embodiment the compound has the formula:

wherein at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is COOR¹³ and each R¹³is independently selected. In some embodiments, the compound has theformula:

Particular embodiments of the invention include methods wherein thevirus is a retrovirus. In some embodiments, the virus is a retrovirusderived from a avian sarcoma and leukosis retroviral group, a mammalianB-type retroviral group, a human T cell leukemia and bovine leukemiaretroviral group, a D-type retroviral group, a murine leukemia-relatedgroup, or a lentivirus group. Often, the virus a lentivirus. Inparticular embodiments, the retrovirus is an HIV-1, an HIV-2, an SIV, aBIV, an ELAV, a Visna, a CaEV, an HTLV-1, a BLV, an MPMV, an MMTV, anRSV, a FeLV, a BaEV, or an SSV retrovirus. Preferably, the retrovirus isHIV-1 or HIV-2.

In the method for inactivating a virus, the contacting of the virus withthe compound can be performed in vivo. In this embodiment, the compoundcan be administered to inhibit the transmission of the virus. Thecompound can be administered intra-vaginally or intra-rectally toinhibit the transmission of the virus. The compound can be administeredto a human as a pharmaceutical formulation. The compound can beadministered to an animal as a veterinary pharmaceutical formulation.The method further comprises contacting the virus with ananti-retroviral agent other than an antimony compound or diphenylcompound. The anti-retroviral agent can be a nucleoside analogue, aprotease inhibitor, or a non-nucleoside reverse trancriptase inhibitor(NNRTI). The nucleoside analogue can be AZT, ddCTP or

DDI. The protease inhibitor can be Indinavir, Saquinavir, or Ritonavirand NNRTI include nevirapine and efavirenz.

In another aspect, the invention also provides a method for inactivatinga virus, wherein the contacting of the virus with the compound can beperformed in vitro. In this embodiment of the method, the contacting ofthe retrovirus with the compound can be performed in a blood product,blood plasma, nutrient media, protein, a pharmaceutical, a cosmetic, asperm or oocyte preparation, cells, cell cultures, bacteria, viruses,food or drink.

In one aspect of the methods of the invention, the compound isadministered to a human as a pharmaceutical formulation. Often thecompound is administered intra-vaginally or intra-rectally to inhibitthe transmission of the virus.

In another embodiment, the compound is administered to an animal as aveterinary pharmaceutical formulation. Preferably, the pharmaceuticalformulation comprises a unit dose of a compound described herein. Often,the pharmaceutical formulation further comprises a pharmaceuticalexcipient.

DETAILED DESCRIPTION

The efficacy of most antiviral agents is limited because it is commonthat, under selection pressure, viruses mutate to drug-resistantstrains. Development of drug resistance is a survival strategyparticularly pronounced amongst retroviruses because of their ability torapidly mutate. Viral structures that are required for viability andreplication are typically considered as good drug targets because theirinactivation cannot be easily overcome by mutation, thus often thesestructure are mutationally intolerant. Furthermore, these structuresmaybe conserved and/or maintained between virus families, groups orgenera, thus providing a common target for the development of antiviralagents or therapies.

HIV-1's nucleocapsid (NC) protein, NCp7, contains two zinc fingersseparated by only seven amino acids (Henderson (1992) J. Virol.66:1856). Both fingers are essential for infectivity (Aldovini (1990) J.Virol. 64:1920; Gorelick (1990) J. Virol. 64:3207). Agents have beenidentified that target this regions, see, e.g., WO97/44064, WO99/65871,U.S. Pat. Nos. 6,001,555 and 6,046,228. The present invention employscompounds comprising an to antimony structure or diphenyl structurewhich bind to the nucleocapsid with high affinity and thereby inhibitinteraction with nucleic acid molecules.

In a first aspect of the invention, there is provided a method ofinhibiting viral replication using antimony compounds having a structureaccording to Formula I:

In Formula I, R¹⁴, R¹⁵ and R¹⁶ are members independently selected fromH, NO₂, Sb(O)(OH)₂, OR¹⁷, SR¹⁷, CN, NR¹⁷R¹⁸, COR¹⁸, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. R¹⁷and R¹⁸ are members independently selected from H, OR¹⁹, C(O)R¹⁹, andNR¹⁹R²⁰. R¹⁹ and R²⁰ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. At least one of R¹⁴, R¹⁵ and R¹⁶ is other than H.

In another aspect of the invention, there is provided a method ofinhibiting viral replication using diphenyl compounds having a structureaccording to Formula II:

In Formula II, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, CN, OR¹¹, COR¹², NR¹¹R¹³, andCONR¹¹R¹³. R¹¹ and R¹³ are members independently selected from H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl. R¹² is a member selected from H, and OR¹³; and X is amember selected from O, S, and NR^(x). R^(x) is a member selected fromH, substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. At least three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, andR¹⁰ are COOR¹³.

In another embodiment, the method of the invention utilizes compoundshaving a structure according to Formula III:

In Formula III, at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is COOR¹³ andeach R¹³ is independently selected.

In another embodiment, the compounds have a structure according toFormula IV:

In Formula IV, X is typically O.

The present invention is directed to the use of aromaticantimony-containing compounds and diphenyl compounds as described hereinto disrupt the association of a viral nucleocapsid protein to nucleicacid. Many of these compounds are known, see, e.g., the National CancerInstitute chemical repository although none have been used as anticancer(or other indication) agents prior to the present application. Chemicalstructural information on some of the claimed compound is available viathe DTP website: http://dtp.nci.nih.gov. Methods of synthesizing suchchemicals are known to those of skill in the art. Moreover, newcompounds according to Formula I, II, III, or IV that inhibit viralreplication as described herein can be synthesized using techniquesreadily apparent to those of skill in the art. For example, antimonycompounds of the invention may be prepared in accordance with thefollowing references: Doak et al., Preparation of stibonic acids by theScheller reaction. J/ Am. Chem. Soc. 1946, 68, 1987-1989. Enger &Sweeting The preparation of aromatic stibonic acids of certainbenzenesulfonamides. J. Am. Chem. Soc. 1948, 70, 2977-2979. Reutov &Ptitsyna, Synthesis of organoantimony compounds through double diazoniumsalts. Doklady Akad. Nauk S.S.S.R. 1951, 79, 819-821. The diphenylcompounds maybe prepared, for example, in accordance with the following:Kobata et al. Manufacture of polyesters. Japan. (1978), 4pp. CODEN:JAXXAD JP 53023357 19780714 Showa. CAN 89:164360 AN 1978:564360 CAPLUS.

Definitions

To facilitate understanding the invention, a number of terms are definedbelow.

An “aromatic antimony-containing compound” or “antimony compound” asused herein refers to a compound having a structure corresponding tothat set out in Formula I. This formula defines a three-dimensionalphamacophore which interacts with the nucleocapsid protein by forming atleast one hydrogen bond. Other antimony compounds to exploit the presentand other hydrogen bonding modalities will be apparent to those of skillin the art.

A “diphenyl compound” as used herein refers to a compound having astructure corresponding to that set out in Formulas II, III, and IV.These formulas define a three-dimensional phamacophore which interactswith the nucleocapsid protein by forming at least one hydrogen bond.Other diphenyl compounds to exploit the present and other hydrogenbonding modalities will be apparent to those of skill in the art.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (up to three rings) which are fused together or linked covalently.The term “heteroaryl” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers.

The term “alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups which are limited to hydrocarbon groups aretermed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from at least one heteroatom selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S and Si maybe placed at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms 30 canalso occupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂R—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NRW″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′₂)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

Similar to the substituents described for the aryl radical, substituentsfor the alkyl and heteroalkyl radicals (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, and heteroalkyl,unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstitutedalkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkyl groups. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. When R′and R″ are attached to the same nitrogen atom, they can be combined withthe nitrogen atom to form a 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those-derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al, “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

“Contacting” refers to the act of bringing components of a reaction intoadequate proximity such that the reaction can occur. More particularly,as used herein, the term “contacting” can be used interchangeably withthe following: combined with, added to, mixed with, passed over, flowedover, etc.

As used herein, the term “Gag-Pol protein” refers to the polyproteintranslation product of HIV-1 or other retroviruses, as described, e.g.,by Fehrmann (1 997) Virology 235:352359; Jacks (1988) Nature331:280-283. The “Gag protein” is processed by a viral protease to yieldmature viral proteins, see, e.g., Humphrey (1997) Antimicrob. AgentsChemother. 41:1017-1023; Karacostas (1993) Virology 193:661-671.

The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

As used herein, “isolated,” when referring to a molecule or composition,such as, for example, an antimony compound or diphenyl compound of theinvention, an antimony compound- or diphenyl compound-complexedpolypeptide or virus, or an antimony compound- or diphenylcompound-inactivated virus, means that the molecule or composition isseparated from at least one other compound, such as a protein, othernucleic acids (e.g., RNAs), or other contaminants with which it isassociated in vivo or in its naturally occurring state. Thus, acompound, polypeptide or virion is considered isolated when it has beenisolated from any other component with which it is naturally associated,e.g., cell membrane, as in a cell extract, serum, and the like. Anisolated composition can, however, also be substantially pure. Anisolated composition can be in a homogeneous state and can be in a dryor an aqueous solution. Purity and homogeneity can be determined, forexample, using analytical chemistry techniques such as polyacrylamidegel electrophoresis (SDS-PAGE) or high performance liquid chromatography(HPLC).

As used herein, the term “nucleocapsid protein” or “NC protein” refersto the retroviral nucleocapsid protein, which is an integral part of thevirion nucleocapsid, where it coats the dimeric RNA genome, as describedby, e.g., Huang (1997) J. Virol. 71:4378-4384; Lapadat-Tapolsky (1997)J. Mol. Biol. 268:250-260. HIV-1's nucleocapsid protein is termed“NCp7,” see also Demene (1994) Biochemistry 33:11707-11716.

All NC proteins of the Oncovirinae and Lentivirinae subfamilies ofRetroviridae contain sequences of 14 amino acids with 4 invariantresidues, Cys(X)₂Cys(X)₄His(X)₄Cys, (L. E. Henderson et al. J. Biol.Chem. 256, 8400 (1981)) which chelate zinc through histidine imidazoleand cysteine thiolates with a K_(d) less than 10⁻¹³ (J. M. Berg, Science232, 485 (1986); J. W. Bess, Jr., et al., J. Virol. 66, 840 (1992); M.R. Chance et al., Proc. Natl. Acad. Sci. U.S.A. 89, 10041 (1992); T. L.South and M. F. Summers, Adv. Inorg. Biochem. 8, 199 (1990); T. L.South, et al., Biochem. Pharmacol. 40, 123 (1990)). These structures arereferred to as retroviral CCHC zinc fingers, and are one of the mosthighly conserved features of retroviruses. Examples of retroviruseswhich possess at least one CCHC type zinc finger per nucleocapsidprotein include, but are not limited to, HIV-1, HIV-2, SIV, BIV, EIAV,Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, FeLV, BaEV, and SSV.

The term “retrovirus” as used herein refers to viruses of theRetroviridae family, which typically have ssRNA transcribed by reversetranscriptase, as defined by, e.g., P. K. Vogt, “Historical introductionto the general properties of retroviruses,” in Retroviruses, eds. J. M.Coffin, S. H. Hughes and H. E. Varmus, Cold Spring Harbor LaboratoryPress, 1997, pp 1-26; Murphy et al. (eds.) Archives ofVirology/Supplement 10, 586 pp (1995) Springer Verlag, Wien, N.Y.; andthe web site for the Committee on International Taxonomy of Viruses,Virology Division of the International Union of Microbiology Society athttp://www.ncbi.nlm.nih.gov/ICTV/ for viral classification and taxonomy.Retroviridae family members containing zinc finger motif-containingpolypeptides and whose replication can be inhibited by the antimonycompounds of the invention include avian sarcoma and leukosisretroviruses (alpharetroviruses), mammalian B-type retroviruses(betaretrovirus) (e.g., mouse mammary tumor virus), human T cellleukemia and bovine leukemia retroviruses (deltaretroviruses) (e.g.,human T-lymphotropic virus 1), murine leukemia-related group(gammaretroviruses), D-type retroviruses (epsilonretrovirus) (e.g.Mason-Pfizer monkey virus), and Lentiviruses. Lentiviruses includebovine, equine, feline, ovine/caprine, and primate lentivirus groups,such as human immunodeficiency virus 1 (HIV-1). Examples of particularspecies of viruses whose replicative capacity is affected by theantimony compounds of the invention include HIV-1, HIV-2, SIV, BIV,ELAV, Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, andSSV retrovirus.

Other viruses that include proteins with zinc finger domains thatinteract with nucleic acid can also be targeted with the antimonycompounds as described herein. For example, human papilloma virus E6 andE7 proteins contain zinc finger domains that can be targets for bindingby this series of compounds (Beerheide et al. J Natl Cancer Inst 91:1211-20, 1999). Likewise, the hepatitis C virus genome codes -for zincfinger-containing proteins that can be targeted with the antimonycompounds.

Determining Anti-Viral Activity of the Antimony and Diphenyl Compounds

In determining the anti-viral activity of an antimony compound ordiphenyl compound of the invention, the ability of the compound to bindto a viral nucleocapsid protein is often evaluated. The assessment ofbinding affinity can be determined using techniques known by one orordinary skill in the art (see, e.g., WO97/44064). For exampletryptophan fluorescence quenching or other binding assays such as theBIAcore chip technology can be used in order to determine the bindingaffinity of an antimony or diphenyl compound for the viral zincfinger-containing protein. Examples of these procedures are furtherprovided in Example 1. As appreciated by one of skill, other assayprocedures can be used to provide an equivalent assessment for bindingof compounds to any viral nucleocapsid protein. In some embodiments,binding affinity is assessed and compounds that bind to the nucleocapsidat a K_(D) of less than 200 μM, determined, e.g., using a solutioninhibition assay as described in the Examples, are then analyzed foranti-viral activity in vitro.

An antimony compound or diphenyl compound is within the scope of theinvention if it displays any antiviral activity (i.e., any ability todecrease the cytopathic effect or diminish the transmission of or thereplicative capacity of a virus). The antiviral activity can bedetermined empirically by clinical observation or objectively using anyin vivo or in vitro test or assay, e.g., the XTT cytoprotection assay(described herein), measuring Tat-induced activity (as in theHeLa-CD4-LTR-beta-gal (MAGI cells) assay and detecting Tat-inducedbeta-galactosidase activity, see, e.g., Tokunaga (1998) J. Virol.72:6257-6259), and the like. An antimony or diphenyl compound with anydegree of measurable antiviral activity is within the scope of theinvention.

One exemplary means to determine antiviral activity is with CEM-SS cellsand virus (e.g., HIV-1_(RF)) (MOI=0.01) using the XTT(2,3-bis[2-methoxy- 4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) cytoprotection assay(see, e.g., Weislow, et al, J. Natl. Canc. Inst. 81: 577-586, 1989; RicePNAS 90:9721-9724, 1993; and Rice Antimicrob. Agents Chemother.41:419-426, 1997). Briefly, cells are infected with HIV-1_(RF) (or othervirus to be tested) in the presence of various dilutions of testcompounds (antimony or diphenyl compounds and controls). The culturesare incubated for seven days. During this time control cultures withoutprotective compounds (i.e., compounds with anti-viral activity)replicate virus, induce syncytia, and result in about 90% cell death.The cell death is measured by XTT dye reduction. XTT is a solubletetrazolium dye that measures mitochondrial energy output, similar toMTT. Positive controls using dextran sulfate (an attachment inhibitor)or 3′-Azido -2′-3′-dideoxythymidine, AZT (a reverse transcriptaseinhibitor) are added to each assay. Individual assays are done induplicate using a sister plate method.

Effective antiviral concentrations providing 50% cytoprotection (EC₅₀),and cellular growth inhibitory concentrations causing 50% cytotoxicity(IC₅₀) are calculated.

Alternatively, any virus can be grown in culture, or in an in vivo(animal) model, in the presence or absence of an antimony compound or anantimony-containing pharmaceutical formulation to test for anti-viral,viral transmission-inhibiting activity and efficacy. Any virus, assay oranimal model which would be apparent to one of skill upon review of thisdisclosure can be used, see, e.g., Lu (1997) Crit. Rev. Oncog.8:273-291; Neildez (1998) Virology 243:12-20; Geretti (1998) J. Gen.Virol. 79:415-421; Mohri (1998) Science 279:1223-1227; Lee (1998) Proc.Natl. Acad. Sci. USA 95:939-944; Schwiebert (1998) AIDS Res. Hum.Retroviruses 14:269-274.

For in vitro assays, any measurable decrease in the viral load of aculture grown in the presence of an antimony or diphenyl test compoundas compared to a positive or negative control compound is indicative ofan anti-viral, transmission-inhibiting effect. Typically, at least a 30%reduction in viral load observed, generally, between 10% and 99%. Asdiscussed in definition section, above, any relevant criteria can beused to evaluate the antiviral efficacy of an antimony composition orantimony-containing formulation.

Cloning and Expression of Viral Nucleocapsid Proteins

The antimony and diphenyl compounds of the invention of the inventionprevent the binding of viral nucleocapsid protein to nucleic acids. Inorder to identify such compounds, the ability of antimony or diphenylcompounds to bind to the viral nucleocapsid is assessed in a bindingassay using the targeted nucleocapsid protein, often a retroviralprotein. The viral nucleocapsid proteins to detect the binding andantiviral activity of the antimony and diphenyl compounds are typicallyproduced using-recombinant technology. General laboratory procedures forthe cloning and expression of nucleocapsid proteins can be found, e.g.,current editions of Sambrook, et al., Molecular Cloning A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.Greene Publishing and Wiley-Interscience, N.Y. (1987). Sequences andsources of nucleocapsid proteins, including nucleic acids, proteins andviral sources, are publicly available, for example, through electronicdatabases, such as, e.g., The National Center for BiotechnologyInformation at http://www.ncbi.nlm.nih.gov/Entrez/, or, The NationalLibrary of Medicine at http:/fwww.ncbi.nlm.nih.gov/PubMed/.

In general, the DNA encoding the polypeptide or peptide of interest arefirst cloned or isolated in a form suitable for ligation into anexpression vector. After ligation, the vectors containing the DNAfragments or inserts are introduced into a suitable host cell forexpression of the recombinant polypeptides. The polypeptides are thenisolated from the host cells. The nucleic acids may be present intransformed or transfected whole cells, in a transformed or transfectedcell lysate, or in a partially purified or substantially pure form.Techniques for nucleic acid manipulation of genes encoding zincfinger-containing proteins, such as subcloning nucleic acid sequencesinto expression vectors, labeling probes, DNA hybridization, and thelike are described, e.g., in Sambrook and Ausubel, supra.

Once the DNAs are isolated and cloned, the desired polypeptides areexpressed in a recombinantly engineered cell such as bacteria, yeast,insect, or mammalian cells. It is expected that those of skill in theart are knowledgeable in the numerous expression systems available forexpression of the recombinantly produced proteins. No attempt todescribe in detail the various methods known for the expression ofproteins in prokaryotes or eukaryotes will be made. In brief summary,the expression of natural or synthetic nucleic acids encodingpolypeptides will typically be achieved by operably linking the DNA orcDNA to a promoter (which is either constitutive or inducible), followedby incorporation into an expression vector. The vectors can be suitablefor replication and integration in either prokaryotes, or eukaryotes.Typical expression vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the DNA encoding recombinant polypeptides. Toobtain high level expression of a cloned gene, it is desirable toconstruct expression plasmids which contain, at the minimum, a promoterto direct transcription, a ribosome binding site for translationalinitiation, and a transcription/translation terminator.

Viricidal Activity of Antimony and Diphenyl Compounds

In another aspect, the invention also provides a method of using acomposition comprising a bio-organic or other material and an amount ofan antimony compound of the invention effective to inactivate any virus(susceptible to inactivation by an antimonycompound) which is or maycontaminate the material. The material can be bio-organic, such as,e.g., blood plasma, nutrient media, protein, a pharmaceutical, acosmetic, a sperm or oocyte preparation, cells, cell cultures, bacteria,viruses, foods, drinks. They can be surgical or other medical materials,such as, e.g., implant materials or implantable devices (e.g., plastics,artificial heart valves or joints, collagens), medical materials (e.g.,tubing for catheterization, intubation, [Vs] and containers (e.g., bloodbags, storage containers), and the like. Alternatively, an antimonycompound of the invention can be in the form of a composition which isapplied to any of the above materials as a viricidal reagent and removedbefore the material's use. The viricidal composition can contain amixture of different antimony or diphenyl compounds of the invention invarying amounts. For example, antimony compounds can be added to cellcultures to reduce the likelihood of viral contamination, providingadded safety for the laboratory workers.

Antimony and Diphenyl Compounds as Pharmaceutical Formulations

The invention also provides pharmaceutical formulations comprising theantimony compounds of the invention. These antimony compounds are usedin pharmaceutical compositions that are useful for administration tomammals, particularly humans, for the treatment of viral, especiallyretroviral, infections.

The compounds of the invention can be formulated as pharmaceuticals foradministration in a variety of ways. Typical routes of administrationinclude both enteral and parenteral. These include, e.g., withoutlimitation, subcutaneous, intramuscular, intravenous, intraperitoneal,intramedullary, intrapericardiac, intrabursal, oral, sublingual, ocular,nasal, topical, transdermal, transmucosal, or rectal. The mode ofadministration can be, e.g., via swallowing, inhalation, injection ortopical application to a surface (e.g., eyes, mucous membrane, skin).Particular formulations typically are appropriate for specific modes ofadministration. Various contemplated formulations include, e.g., aqueoussolution, solid, aerosol, liposomal and transdermal formulations.Details on techniques for formulation and administration are welldescribed in the scientific and patent literature, see, e.g., the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton Pa.).

Aqueous Solutions for Enteral, Parenteral Or Transmucosal Administration

Examples of aqueous solutions that can be used in formulations forenteral, parenteral or transmucosal drug delivery include, e.g., water,saline, phosphate buffered saline, Hank's solution, Ringer's solution,dextrose/saline, glucose solutions and the like. The formulations cancontain pharmaceutically acceptable auxiliary substances to enhancestability, deliverability or solubility, such as buffering agents,tonicity adjusting agents, wetting agents, detergents and the like.Additives can also include additional active ingredients such asbactericidal agents, or stabilizers. For example, the solution cancontain sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate or triethanolamineoleate. These compositions can be sterilized by conventional, well-knownsterilization techniques, or can be sterile filtered. The resultingaqueous solutions can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration.

Aqueous solutions are appropriate for injection and, in particular, forintravenous injection. The intravenous solution can include detergentsand emulsifiers such as lipids. Aqueous solutions also are useful forenteral administration as tonics and administration to mucous or othermembranes as, e.g., nose or eye drops. The composition can contain anantimony compound or diphenyl compound in an amount of about 1 mg/ml to100 mg/ml, more preferably about 10 mg/ml to about 50 mg/ml.

Formulations for Enteral or Transdermal Delivery

Solid formulations can be used for enteral administration. They can beformulated as, e.g., pills, tablets, powders or capsules. For solidcompositions, conventional nontoxic solid carriers can be used whichinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10%-95% of activeingredient.

A non-solid formulation can also be used for enteral (oral)administration. The carrier can be selected from various oils includingthose of petroleum, animal, vegetable or synthetic origin, e.g., peanutoil, soybean oil, mineral oil, sesame oil, and the like. See Sanchez, etal., U.S. Pat. No. 5,494,936. Suitable pharmaceutical excipients includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skin milk, glycerol, propyleneglycol, water, ethanol, and the like. Nonionic block copolymerssynthesized from ethylene oxide and propylene oxide can also bepharmaceutical excipients; copolymers of this type can act asemulsifying, wetting, thickening, stabilizing, and dispersing agents,see, e.g., Newman (1998) Crit. Rev. Ther. Drug Carrier Syst. 15:89-142.

A unit dose form, such as a tablet, can be between about 50 mg/unit toabout 2 grams/unit, preferably between about 100 mg/unit to about 1gram/unit.

Topical Administration: Transdermal/Transmucosal Delivery

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, e.g., for transmucosal administration, bile salts and fusidicacid derivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be through nasal sprays, forexample, or using suppositories.

For topical administration, the agents are formulated into ointments,creams, salves, powders and gels. In one embodiment, the transdermaldelivery agent can be DMSO. Transdermal delivery systems can alsoinclude, e.g., patches.

The antimony or diphenyl compounds can also be administered in sustaineddelivery or sustained release mechanisms, which can deliver theformulation internally. For example, biodegradeable microspheres orcapsules or other biodegradeable polymer configurations capable ofsustained delivery of a composition can be included in the formulationsof the invention (see, e.g., Putney (1998) Nat. Biotechnol. 16:153-157).

Formulation Delivery by Inhalation

For inhalation, the antimony or diphenyl compound formulation can bedelivered using any system known in the art, including dry powderaerosols, liquids delivery systems, air jet nebulizers, propellantsystems, and the like. See, e.g., Patton (1998) Biotechniques16:141-143; inhalation delivery systems by, e.g., Dura Pharmaceuticals(San Diego, Calif.), Aradigrn (Hayward, Calif.), Aerogen (Santa Clara,Calif.), Inhale Therapeutic Systems (San Carlos, Calif.), and the like.

For example, the pharmaceutical formulation can be administered in theform of an aerosol or mist. For aerosol administration, the formulationcan be supplied in finely divided form along with a surfactant andpropellant. The surfactant preferably is soluble in the propellant.Representative of such agents are the esters or partial esters of fattyacids containing from 6 to 22 carbon atoms, such as caproic, octanoic,lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleicacids with an aliphatic polyhydric alcohol or its cyclic anhydride suchas, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, andthe polyoxyethylene and polyoxypropylene derivatives of these esters.Mixed esters, such as mixed or natural glycerides can be employed. Thesurfactant can constitute 0.1%-20% by weight of the composition,preferably 0.25%-5%. The balance of the formulation is ordinarilypropellant. Liquefied propellants are typically gases at ambientconditions, and are condensed under pressure. Among suitable liquefiedpropellants are the lower alkanes containing up to 5 carbons, such asbutane and propane; and preferably fluorinated or fluorochlorinatedalkanes. Mixtures of the above can also be employed. In producing theaerosol, a container equipped with a suitable valve is filled with theappropriate propellant, containing the finely divided compounds andsurfactant. The ingredients are thus maintained at an elevated pressureuntil released by action of the valve. See, e.g., Edwards (1997) Science276:1868-1871.

A nebulizer or aerosolizer device for administering antimony or diphenylcompounds of this invention typically delivers an inhaled dose of about1 mg/m³ to about 50 mg/m³.

Delivery by inhalation is particular effective for delivery torespiratory tissues for the treatment of respiratory conditionsincluding an inflammatory component.

Other Formulations

In preparing pharmaceuticals of the present invention, a variety offormulation modifications can be used and manipulated to alterpharmacokinetics and biodistribution. A number of methods for alteringpharmacokinetics and biodistribution are known to one of ordinary skillin the art. For a general discussion of pharmacokinetics, See,Remington's Pharmaceutical Sciences, supra, Chapters 37-39.

Administration

The antimony or diphenyl compounds of the invention are used in thetreatment and prevention of viral infection, particularly, retroviralinfections. The amount of the compound adequate to accomplish this isdefined as a “therapeutically effective dose.” The dosage schedule andamounts effective for this use, i.e., the “dosing regimen,” will dependupon a variety of factors, including frequency of dosing, the stage ofthe disease or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age and the like. In calculating the dosage regimen for a patient, themode of administration also is taken into consideration.

The dosage regimen must also take into consideration thepharmacokinetics, i.e., the antimony or diphenyl compound's rate ofabsorption, bioavailability, metabolism, clearance, and the like (see.e.g.; the latest Remington's edition, supra).

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical= formulations shouldpro-vide a quantity of an antimony compound or diphenyl compoundsufficient to treat the patient effectively. The total effective amountof an antimony compound or diphenyl compound of the present inventioncan be administered to a subject as a single dose, either as a bolus orby infusion over a relatively short period of time, or can beadministered using a fractionated treatment protocol, in which themultiple doses are administered over a more prolonged period of time.One skilled in the art would know that the concentration of an antimonycompound or diphenyl compound of the present invention required toobtain an effective dose in a subject depends on many factors including,e.g., the pharmacokinetics of the prodrug and of its hydrolysis product,the age and general health of the subject, the route of administration,the number of treatments to be administered and the judgment of theprescribing physician. In view of these factors, the skilled artisanwould adjust the dose so as to provide an effective dose for aparticular use.

As appreciated by one of skill in the art, the antimony or diphenylcompounds can be used in conjunction with other therapies used for thetreatment of viral infection. For example, in HIV-1 infection anantimony compound or diphenyl compound can be used in a therapeuticregimen that includes nucleoside analog therapy an protease inhibitortherapy.

Vaccine Formulations Comprising the Compounds of the Invention

In another aspect, the invention also provides an isolated andinactivated virus, where the virus is inactivated by a method comprisingcontacting the virus with an antimony compound or diphenyl compound ofthe invention, wherein contacting said virus with said compoundinactivates said virus. In one embodiment the isolated and inactivatedvirus further comprises a vaccine formulation.

The antimony compound-complexed or diphenyl compound-complexed,inactivated viruses of the invention are used in vaccine formulationsthat are useful for administration to mammals, particularly humans totreat and generate immunity to of a variety of viral diseases,particularly retroviral infections, such as HIV-1. The vaccineformulations can be given single administrations or a series ofadministrations. When given as a series, inoculations subsequent to theinitial administration are given to boost the immune response and aretypically referred to as booster inoculations.

The vaccines of the invention contain as an active ingredient animmunogenically effective amount of an antimondy compound-complexed ordiphenyl compound-complexed, inactivated, virus. Useful carriers arewell known in the art, and include, e.g., thyroglobulin, albumins suchas human serum albumin, tetanus toxoid, polyamino acids such aspoly(D-lysine: D-glutamic acid), influenza, hepatitis B virus coreprotein, hepatitis B virus recombinant vaccine and the like. Thevaccines can also contain a physiologically tolerable (acceptable)diluent such as water, phosphate buffered saline, or saline, and furthertypically include an adjuvant. Adjuvants such as incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum are alsoadvantageously used to boost an immune response.

Uses of Compound-Inactivated Viruses and Compound-Complexed Proteins

In addition to uses as vaccines, antimony compound-inactivated ordiphenyl compound-inactivated viruses and antimony compound-complexed ordiphenyl-complexed viral proteins have a variety of uses. For example,antimony compound-complexed or diphenyl compound-complexed viralproteins or compound-inactivated viruses can be used as reagents for thedetection of corresponding anti-viral antibodies or as crystallizationreagents for X-ray crystallization analysis or other ultrastructuralstudies, see, e.g., Yamashita (1998) J. Mol. Biol. 278:609-615; Wu(1998) Biochemistry 37:4518-4526.

Kits

In an additional aspect, the present invention provides kits embodyingthe methods herein. Kits of the invention optionally comprise one ormore of the following: (1) a diphenyl or antimony component as describedherein; (2) instructions for practicing the methods described herein,and/or for using the antimony or diphenyl component; (3) one or moreassay component; (4) a container for holding the antimony or diphenylcompound, assay components, or apparatus components useful formanipulating the antimony compound or diphenyl compound or practicingthe methods herein, and, (5) packaging materials.

In a further aspect, the present invention provides for the use of anycompound, kit, or kit component herein, for the practice of any methodor assay herein, and/or for the use of any apparatus or kit to practiceany assay or method herein.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are intended neither to limit or define the invention in any manner.

EXAMPLES Example 1 Binding of Antimony Compounds and Diphenyl Compoundsto Viral Nucleocapsid Protein

High-Throughput Screen

A high throughput screen was used to detect small molecules thatdisrupted the association of viral NC-p7 protein with anoligonucleotide. The assay was performed using a Tomtec Quadra roboticsystem. NC protein was immobilized on Costar (Corning, N.Y.) high-bindpolystyrene 96 well plates by incubating 100 μl 250 nM NC-p7 inphosphate buffered saline (PBS)/10 μM zinc chloride/10 mMβ-mercaptoethanol/0.05% Tween overnight at 4° C. 200 μl of 2% BSA inPBS/10 μM zinc chloride/10 mM β-mercaptoethanol/0.05% Tween was addedand incubated at room temperature for 1-2 hours in order to block theplates. The plates were then washed 2 times with 200 μl PBS/10 μM zincchloride/10 mM β-mercaptoethanol/0.05% Tween using a Titertek M96V platewasher and stored overnight at 4° C. 5 nM biotinylated 28 baseoligonucleotide (5′ GACTTGTGGAAAATCTCTAGCAGTGCAT 3′) in PBS/10 μM zincchloride/10 mM β-mercaptoethanot/0.05% Tween was added to each wellfollowed by 10 μM of test compound (in 20% dimethyl sulphoxide) from theDiversity Set and allowed to incubate at room temperature for 1 hour.Plates were then washed 3 times with 200 μl PBS/0.05% Tween. Binding ofthe biotinylated 28 mer was measured by adding 100 μl 1:20000 dilutionof nutravidin-horse radish peroxidase (stock 0.8 mg/ml, from PierceChemical Co. Rockford, Ill.) and incubating at room temperature for 1hour. The plates were washed 3 times with 200 μl PBS/0.05% Tween. Plateswere developed by adding 100 μl of Supersignal (Pierce Chemical Co.Rockford, Ill.) and bioluminescence was measured in a Wallac Victorplate reader. Each plate had eight positive (5 mM EDTA in PBS) and eightnegative (20% DMSO alone) controls. The hit threshold was set at 100%inhibition. Active compounds that were identified included NSC 13778,NSC 13746, NSC 13755, and NSC 28620.

Secondary Screen

A secondary screen was used to identify small molecules that disrupt invitro assembly of Gag precursor protein into virus-like particles.Artificial viral particles were assembled from recombinant Gag proteinand tRNA as previously described (see, e.g., Campbell & Rein, “In vitroassembly properties of human immunodeficiency virus type 1 Gag proteinlacking the p6 domain” J. Virol. 73: 2270-2279, 1999).

Two series of active compounds were identified that disrupted viralassembly. One series, an aromatic antimony-containing series, is basedon the antimony-containing compound NSC 13778. The other series, adiphenyl series, is based on NSC 28620. Exemplary compounds thatdisrupted viral assembly in the antimony series in addition to NSC 13778included NSC 13746, NSC 13771, NSC 13755, NSC 13759, NSC 13760, NSC13765, and NSC 13793.

A fluorescence polarization assay was used to measure the bindingbetween gag precursor protein and oligonucleotides. Shortoligonucletoides (d(TG)₄) were used to monitor binding. The activity ofthe compounds toward gag precursor protein was assessed using a BIAcoreosolution inhibition assay (e.g., Christensen et al., Anal Biochem249:153-164, 1997; Fivash et al., Curr. Opin. Biotechnol. 9:97-101,1998; Fisher et al., J. Virol. 72:1902-1909, 1998). Gag precursorprotein and a d(TG)₄ oligonucleotides immobilized on a flow surface wereincubated to allow formation of a complex. Increased amounts of thecandidate compounds were then added to disrupt the complex. Dissociationconstant were then calculated. Those compounds that were most effectiveat disrupting the gag precursor protein-d(TG₄) complex were also mostactive in particle disassembly.

The fluorescence polarization assays was repeated substitutingnucleocapsid protein for gag precursor protein in forming a complex withd(TG)₄. Generally, compounds that were most effective in disrupting thegag-d(TG)₄ complex were also the most active toward thenucleocapsid-d(TG₄) complex.

To demonstrate the specificity of the compounds, a third fluorescencepolarization assays was performed that assess the ability of thecompounds to disrupt a complex between the tryptophan repressor proteinand its cognate DNA. No compounds exhibited any activity up to 100 μM.

These experiments demonstrate that the compounds identified specificallyinterfered with the interaction between the nucleocapsid domain of gagprecursor protein and the nucleic acid.

Cell-based Anti-HIV Activity

Cell based anti-HIV screening was performed in CEM-SS cells, using anXTT-cytoprotection assay (see, e.g., Weislow, et al, J. Natl. Canc.Inst. 81: 577-586, 1989; Rice PNAS 90:9721-9724, 1993; and RiceAntimicrob. Agents Chemother. 41:419-426, 1997)). Compounds were rankedas active (80-100% protection from HIV infection), moderate (50-79%protection) and inactive (0-49% protection). Generally, samples that hadlower Kd's, i.e., bound tightly, were found to be active in the cellbased assay. These include the diphenyl compound NSC 28620; and theantimony series compounds NSC 13778, NSC 13746, NSC 13755, NSC 13759,NSC 13760, and NSC 13765. NSC 13782 also exhibited activity in thisassay.

Selected compounds were also tested for the ability to inhibit Moloneymurine leukemia virus, which is a member of the gamma retrovirus familyof retroviruses. The drugs were tested by performing the S+L− focusassay (Bassin et al., Nature 229:564-566, 1971), which is equivalent toa plaque assay, in the presence of the compound. None of the compoundsexhibited an ability to inhibit murine leukemia virus replication.

Experiments with radio-labelled NSC 13778 also provided evidence forenergy-dependent uptake of the compound in CEM-SS Leukemia cells.Further, exemplary experiments in mice showed that NSC 13778 istolerated in vivo.

A summary of exemplary compound activity is provided in Table 1. TABLE 1EC₅₀ (μM) disassembly p7 p24 NCS # in vitro VIP in vivo binding XTTassay 13778 3.8 A A A 0.11 13755 6.15 A M A 0.21 13759 1.5 A M A 0.1913760 1.15 A M A 0.29 13765 3.75 A I A 0.41 13771 5.5 A I I 0.2 137933.25 A A I 0.14 13746 10.0 A A A not testedExemplary Compounds

The series of compound comprising dihydroxy(oxido)stibino benzenederivatives exhibited several features that were associated withenhanced activity. The proper orientation of the substituents was onefactor in obtaining enhanced activity. Typically, the meta orientationof the stibino group relative to the carbonyl group conferred optimalactivity.

Exemplary Compounds in the Aromatic Stibonic Acid Family havingAnti-Viral Activity:

The following compounds have activity in at least one of the assays ofTable 1, i.e., an in vivo disassembly VIP assay, a p7 binding assay, anXTT assay, or a p24 assay.

Exemplary diphenyl compound having antiviral activity:

Thus, these examples showed that antimony and diphenyl compounds havebeen identified that disrupt in vitro assembled Gag particles. Thesecompounds also disrupted a complex of NC-p7 or Gag precursor protein(ˆp6) and d(TG)4, which correlates with the particle disassemblyactivity. Further, host cells were protected from the cytopathic effectsof HIV-1 at non-toxic doses of compounds.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

1. A method of inhibiting replication of a virus, said methodcomprising: contacting a nucleocapsid protein of the virus with acompound having the formula:

wherein R¹⁴, R¹⁵ and R¹⁶ are members independently selected from H, NO₂,Sb(O)(OH)₂, OR¹⁷, SR¹⁷, CN, NR¹⁷R¹⁸, COR¹⁸, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl wherein R¹⁷ and R¹⁸are members independently selected from H, OR¹⁹, C(O)R¹⁹, and NR¹⁹R²⁰wherein R¹⁹ and R²⁰ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl, with the proviso that at least one of R¹⁴, R¹⁵ and R¹⁶ isother than H.
 2. The method according to claim 1, wherein at least oneof R¹⁴, R¹⁵ and R¹⁶ comprises a member selected from carboxylic acid,carboxylic acid ester, and carboxylic acid amide.
 3. A method ofinhibiting replication of a virus, said method comprising: contacting anucleocapsid protein of the virus with a compound having the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, CN, OR¹¹, COR¹², NR¹¹R¹³, andCONR¹¹R¹³ wherein R¹¹ and R13 are members independently selected from H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl; R¹² is a member selected from H, and OR¹³; and X is amember selected from O, S, and NR^(x) wherein R^(x) is a member selectedfrom H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl, with the proviso that at least three of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are COOR³.
 4. The method ofclaim 3, wherein the compound has the formula:

wherein at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is COOR¹³ and each R¹³is independently selected.
 5. The method of claim 4, wherein thecompounds has the formula:


6. The method of claim 5, wherein X is O.
 7. The method according toclaim 1 or claim 4, wherein the virus is a lentivirus.
 8. The method ofclaim 7, wherein the lentivirus is an HIV-1, an HIV-2, or an HTLV-1. 9.The method according to claim 1 or claim 4, wherein the contacting stepoccurs in vivo.
 10. The method according to claim 1 or claim 4, whereinthe method further comprises contacting the virus with an anti-viralagent different from the compounds set out in claim
 1. 11. The method ofclaim 10, wherein said anti-viral agent is a anti-retroviral agent thatis a nucleotide analogue or a protease inhibitor.
 12. The method ofclaim 11, wherein said anti-retroviral agent is a nucleotide analogue.13. The method of claim 12, wherein the nucleotides analogue is selectedfrom the group consisting of an AZT, a ddCTP or a DDI analogue.
 14. Themethod of claim 11, wherein the anti-retroviral agent is a proteaseinhibitor.
 15. The method of claim 1 or claim 4, wherein said compoundis administered to a human as a pharmaceutical formulation.
 16. Themethod of claim 15, wherein said compound is administeredintra-vaginally or intra-rectally to inhibit the transmission of thevirus.
 17. The method of claim 15, wherein said compound is administeredto an animal as a veterinary pharmaceutical formulation.
 18. Apharmaceutical formulation comprising a unit dose of a compound set outin claim 1 or claim
 4. 19. The pharmaceutical formulation of claim 18,further comprising a pharmaceutical excipient.